The goal of this project is the determination of the mechanism and result of connections between the microfilament system and the plasma membrane. Previously we had analyzed the system of non-erythroid spectrins as a model for this subcortical network. We now propose to extend our research to the analysis of 36,000 Mr tyrosine kinase substrates termed calpactins. These proteins which exist as monomers or as complexes with a 10,000 Mr light chain interact with the cytoskeletal proteins actin and spectrin and are modulated by Ca++ and phospholipid. We will identify other members of this family using a technique which relies on its ability to interact with actin and phospholipid, and a detailed comparison of the structure and function of these proteins will be made. A detailed analysis of Ca++-binding, including affinity modulators will be undertaken. We will analyze the functional relatedness of the light chain with the S-100 proteins. Monoclonal and polyclonal antibodies will be raised to defined regions (peptides) of calpactin for use in analysis of its function. The tissue and subcellular distribution using Western Blotting and immunofluorescence microscopy. The in vivo target of calpactins will be determined using cross-linking reagents followed by immunoprecipitation. The calpactin heavy and light chain genes will be expressed in bacteria and protein products will be purified and tested for biological activity. We will attempt to identify the region of calpactin needed for Ca++-binding. The effect of tyrosine phosphorylation of calpactin will be tested by determining whether there is any difference in the ability of phosphorylated calpactin to interact with phospholipid, Ca++ or spectrin. These studies will further our understanding of the subcortical filament network and its relationship to cell transformation.
Showing the most recent 10 out of 17 publications
